The complete mitochondrial genome of Pyxicephalus adspersus: high gene rearrangement and phylogenetics of one of the world’s largest frogs Yin-Yin Cai1, Shi-Qi Shen1, Li-Xu Lu1, Kenneth B. Storey2, Dan-Na Yu1,3 and Jia-Yong Zhang1,3 1 College of Chemistry and Life Science, Zhejiang Normal University, Jinhua, Zhejiang, China 2 Department of Biology, Carleton University, Ottawa, ON, Canada 3 Key Lab of Wildlife Biotechnology, Conservation and Utilization of Zhejiang Province, Zhejiang Normal University, Jinhua, Zhejiang, China ABSTRACT The family Pyxicephalidae including two subfamilies (Cacosterninae and Pyxicephalinae) is an ecologically important group of frogs distributed in sub- Saharan Africa. However, its phylogenetic position among the Anura has remained uncertain. The present study determined the complete mitochondrial genome sequence of Pyxicephalus adspersus, the first representative mitochondrial genome from the Pyxicephalinae, and reconstructed the phylogenetic relationships within Ranoidae using 10 mitochondrial protein-coding genes of 59 frog species. The P. adspersus mitochondrial genome showed major gene rearrangement and an exceptionally long length that is not shared with other Ranoidae species. The genome is 24,317 bp in length, and contains 15 protein-coding genes (including extra COX3 and Cyt b genes), four rRNA genes (including extra 12S rRNA and 16S rRNA genes), 29 tRNA genes (including extra tRNALeu (UAG), tRNALeu (UUR), tRNAThr, tRNAPro, tRNAPhe, tRNAVal, tRNAGln genes) and two control regions (CRs). The Dimer- Mitogenome and Tandem duplication and random loss models were used to explain Submitted 29 April 2019 Accepted 22 July 2019 these gene arrangements. Finally, both Bayesian inference and maximum likelihood Published 23 August 2019 analyses supported the conclusion that Pyxicephalidae was monophyletic and that Corresponding authors Pyxicephalidae was the sister clade of (Petropedetidae + Ptychadenidae). Dan-Na Yu, [email protected] Jia-Yong Zhang, [email protected] Subjects Evolutionary Studies, Genomics, Zoology Keywords Pyxicephalidae, Mitogenome, Gene rearrangement, Phylogenetic relationship Academic editor Tony Robillard BACKGROUND Additional Information and Declarations can be found on Anuran mitochondrial (mt) genomes are closed double-stranded DNA molecules with the page 13 lengths varying from 15 to 23 kb (Kakehashi et al., 2013; Zhang et al., 2018a) and typically DOI 10.7717/peerj.7532 encode 37 genes: two ribosomal RNAs (12S and 16S rRNAs), 22 transfer RNAs (tRNAs), Copyright 13 protein-coding genes (PCGs), and the control region (CR) or D-loop region (Boore, 2019 Cai et al. 1999, 2000). The mt genome has several valuable characteristics that have led to its wide Distributed under use as a research tool including limited recombination, maternal inheritance, rapid Creative Commons CC-BY 4.0 evolutionary rate, small size, and relatively conserved gene arrangement (Avise, 1994). Complete or partial mt genomes have been extensively used to test evolutionary How to cite this article Cai Y-Y, Shen S-Q, Lu L-X, Storey KB, Yu D-N, Zhang J-Y. 2019. The complete mitochondrial genome of Pyxicephalus adspersus: high gene rearrangement and phylogenetics of one of the world’s largest frogs. PeerJ 7:e7532 DOI 10.7717/peerj.7532 hypotheses, to study population biogeography and phylogenetic relationships, and to distinguish cryptic species (Boore, 1999; Yu et al., 2015; Zhang et al., 2008). To date, there are 7,977 known species of Anura (Frost, 2019) on Earth, the vast majority of which have not had their mt genomes sequenced and studied. According to Jacob Machado et al. (2018) and Zhang et al. (2018a), there are partial mt genomes for over one hundred species representing 35 families (72 genera) and complete mt genomes for over two hundred species representing 27 families (76 genera) listed in GenBank. Although non-neobatrachian frogs show conservation of the typical mt gene organization, ranoid frogs belonging to the neobatrachia show a variety of gene rearrangements in their mt genomes (Kurabayashi & Sumida, 2013; Zhang et al., 2013). Hence, by increasing the number of sequenced frog mt genomes, we can gain valuable information about mt genome arrangements and improve our understanding of mt genomic phylogenetics and evolution, population genetics (Cai et al., 2018; Cheng et al., 2018; Lin et al., 2014; Ni et al., 2015; Ye et al., 2016) and mt genome expression (Zhang et al., 2019). Some gene rearrangements in the mt genome have been reported in neobatrachians. For example, some species show a rearrangement of the ND5 gene (Alam et al., 2010; Anoop et al., 2017; Kumar et al., 2017; Li et al., 2014a, 2016a; Sano et al., 2004, 2005). A gene rearrangement of ND6 gene has also been reported in several species (Zhang et al., 2018a; Irisarri et al., 2010; Carr et al., 2015; Kakehashi et al., 2013). Two tRNAMet genes have also been found in many species of Ceratobatrachidae, Dicroglossidae, Mantoidae, and Megophryidae (Jiang et al., 2018; Kiran et al., 2017; Kurabayashi et al., 2008; Li et al., 2018a, 2018b; Liang et al., 2016). Mantella madagascariensis (Kurabayashi et al., 2006), Rana kunyuensis (Li et al., 2016a), and Rhacophorus schlegelii (Sano et al., 2005) possess duplicated CRs. By contrast, the tRNAThr gene was lost in Nanorana taihangnica (Chen et al., 2015) and the ATP8 and ND5 genes were lost in Polypedates megacephalus (Zhang et al., 2005). The phylogenetic relationship of Pyxicephalidae was hotly disputed. Pyxicephalidae contains two subfamilies (Cacosterninae and Pyxicephalinae) with a total of 12 genera and 84 recognized species according to molecular and morphological data (Frost et al., 2006). Using mtDNA and morphological data, Van Der Meijden suggested that Pyxicephalinae, which is composed solely of Aubria and Pyxicephalus (Frost et al., 2006), was imbedded within the Discroglossine (Van Der Meijden et al., 2005). By contrast, Wiens et al. (2009) considered Pyxicephalinae as one subfamily of Ranidae. Frost et al. (2006) and Roelants et al. (2007) recovered the monophyly of Pyxicephalidae and Van Der Meijden et al. (2011) and Pyron & Wiens (2011) treated Pyxicephalidae as a valid family. Zhang et al. (2013) supported Pyxicephalidae as a sister clade to Petropedetidae. Bittencourt-Silva et al. (2016) discussed the phylogenetic relationship of the genera in Pyxicephalidae and concluded that Aubria and Pyxicephalus, belonging to Pyxicephalinae, were the basal clade of Pyxicephalidae. Hence, research on the phylogenetic relationships of Pyxicephalus could reveal the monophyly of Pyxicephalidae. The African giant bullfrog (Pyxicephalus adspersus) belongs to the subfamily Pyxicephalinae of the family Pyxicephalidae. It is an explosive-breeding anuran that inhabits arid to subtropical grasslands and savanna across most of southern Africa Cai et al. (2019), PeerJ, DOI 10.7717/peerj.7532 2/19 (Channing, 2001; Minter et al., 2004). It is one of the world’s largest amphibians, with males (unusually larger than females) reaching a length of 225 mm (Wager, 1965) and weights of 1.4–2.0 kg (Channing, DU Preez & Passmore, 1994; Loveridge, 1950). Liu et al. (2013) found Scapharca broughtonii (Mollusca: Bivalvia), which is an enlarged species compared to other species of Scapharca, had largest mitochondrial genomes among the genus Scapharca (Liu et al., 2013). A species with a giant size within an animal group may be associated with a larger mitogenome (Alam et al., 2010; Yu et al., 2012b, 2015; Zhang et al., 2018a). However, no studies in Pyxicephalus adspersus has examined the potential involvement of mt genome evolution. So we wondered whether this giant bullfrog had novel features of its mt genome. In the present study, we sequenced the mt genome of Pyxicephalus adspersus,thefirst representative mt genome of the Pyxicephalinae, to determine if mitochondrial gene rearrangement occurred and to study the phylogenetic relationships of Pyxicephalidae. MATERIALS AND METHODS Animal treatments Pyxicephalus adspersus are not protected by provisions in the laws of the People’s Republic of China on the protection of wildlife. Two samples of Pyxicephalus adspersus were purchased from a pet market and bred in the laboratory of JY Zhang at the College of Life Science, Zhejiang Normal University. Sample acquisition was reviewed, approved and carried out in accordance with the relevant guidelines of the Committee of Animal Research Ethics of Zhejiang Normal University. DNA extraction, PCR and sequencing Total DNA was extracted from the clipped toe of one specimen using a DNeasy Tissue Kit (Qiagen, Hilden, Germany). We amplified 13 overlapping gene fragments by normal polymerase chain reaction (PCR) and long-and-accurate (LA) PCR methods slightly modified from Yu et al. (2015) and Zhang et al. (2013). However, these methods failed to find some mitochondrial genes including ND3, ND5, and tRNAIle, tRNACys, tRNALys, and tRNAArg. We subsequently designed one pair of primers to sequence the ND5 gene (ND5-J: ATRGARGGNCCNACACCWGT; ND5-N: CCCATNTTWCGRATRTCCTGGTC) based to the known mitochondrial gene sequences of 53 Ranidae species from GenBank. After we obtained the ND5 gene fragment, we then designed two pairs of specific primers for ND6 (ND65-J: ACAAGAGCAGAACAATAAGC, ND65-N: TAGAGTGGAGTAAGGCAGAA and ND56-J: ATACAACCGAATTGGAGACA, ND56-N: GGTAAATCAGTGGGTAGGTAT) to sequence the fragment ranging from ND6 to ND5 genes and the fragment ranging from ND5 to ND6 genes, respectively. Surprisingly, when the two fragments from ND6 to